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EP4006167B1 - Composition pour améliorer la performance de test pcr en temps réel, liquide de réaction, utilisation et procédé - Google Patents

Composition pour améliorer la performance de test pcr en temps réel, liquide de réaction, utilisation et procédé Download PDF

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Publication number
EP4006167B1
EP4006167B1 EP20931793.2A EP20931793A EP4006167B1 EP 4006167 B1 EP4006167 B1 EP 4006167B1 EP 20931793 A EP20931793 A EP 20931793A EP 4006167 B1 EP4006167 B1 EP 4006167B1
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composition
present
sample
reaction liquid
detection
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EP4006167A4 (fr
EP4006167C0 (fr
EP4006167A1 (fr
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Lizhong Dai
Bozhi JI
Kang Wu
Jia Liu
Zhongping DENG
Weimin MIAO
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Sansure Biotech Inc
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Sansure Biotech Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present invention belongs to the field of molecular biology detection, in particular to a composition for improving PCR detection performance, a kit, and a method, and more particularly to the improvement of sensitivity, specificity, and interference resistance of fluorescent quantitative PCR.
  • Real-time fluorescent quantitative PCR is a method for measuring the total amount of a product after each polymerase chain reaction (PCR) cycle with a fluorescent chemical in a nucleic acid amplification reaction. It is a method for quantitative analysis of a specific DNA sequence in a sample under test by an internal reference or external reference method.
  • Real-time PCR is real-time detection of a PCR process by fluorescence signals during PCR amplification.
  • the Ct value of a template has a linear relationship with an initial copy number of the template during an exponential period of the PCR amplification, which thus becomes a basis for quantification.
  • Fluorescent indicators for qPCR detection are mainly divided into two categories: one is fluorescent probes, such as Taqman probes and molecular beacon probes; the other is fluorescent dyes that can bind to a double-stranded DNA, such as SYBR Green and EvaGreen.
  • Technologies for improving the detection performance of real-time fluorescent quantitative PCR discussed herein mainly involve improvement of the qPCR detection performance of a fluorescent probe method, especially a Taqman probe method.
  • Chinese Patent CN 1981055 A mentioned the application of a mixture containing a polynucleotide polymerase to improve the stability of a PCR reaction liquid
  • Chinese Patent CN 103409540 A mentioned the use of a novel dye Gelgreen I combined with a Taq enzyme to improve and optimize the amplification efficiency of qualitative PCR
  • Chinese Patent CN 1464070 A mentioned the use of gold nanoparticles with different particle sizes as an identification amplifier of a DNA detector to improve the detection sensitivity in DNA detection.
  • WO 2018/113351 discloses methods for detecting nucleic acids with the aim to improve the results based on additives placed in the amplification reaction solution.
  • the present invention provides a composition for improving qPCR detection performance, the composition comprising: bovine serum albumin, sorbitol, ammonium sulfate, formamide, and tetramethylammonium chloride, and at least one of dithiothreitol and betaine.
  • the composition comprises bovine serum albumin, dithiothreitol, sorbitol, ammonium sulfate, formamide, and tetramethylammonium chloride.
  • the composition comprises bovine serum albumin, sorbitol, betaine, ammonium sulfate, formamide, and tetramethylammonium chloride.
  • the composition comprises bovine serum albumin, dithiothreitol, sorbitol, betaine, ammonium sulfate, formamide, and tetramethylammonium chloride.
  • the bovine serum albumin has a final concentration of 10-150 ⁇ g/mL, preferably 80-120 ⁇ g/mL in the qPCR reaction liquid, for example, 70 ⁇ g/mL, 75 ⁇ g/mL, 80 ⁇ g/mL, 85 ⁇ g/mL, or 90 ⁇ g/mL, more preferably, a final concentration of 80 ⁇ g/mL.
  • the dithiothreitol has a final concentration of 1-10 mM, preferably 2-8 mM in the qPCR reaction liquid, for example, 2 mM, 4 mM, 6 mM, or 8 mM, more preferably, a final concentration of 3 mM.
  • the sorbitol has a final concentration of 1-10 w/v%, preferably 4-6 w/v% in the qPCR reaction liquid, for example, 4 w/v%, 5 w/v%, or 6 w/v%, more preferably, a final concentration of 4 w/v%.
  • the betaine has a final concentration of 0.5-4 mol/L, preferably 0.6-1 mol/L in the qPCR reaction liquid, for example, 0.6 mol/L, 0.7 mol/L, 0.8 mol/L, or 0.9 mol/L, more preferably, a final concentration of 0.8 mol/L.
  • the ammonium sulfate has a final concentration of 2-50 mM, preferably 8-15 mM in the qPCR reaction liquid, for example, 8 mM, 9 mM, 10 mM, 11 mM, or 12 mM, more preferably, a final concentration of 10 mM.
  • the formamide has a final concentration of 0.1-10 v/v%, preferably 0.5-5 v/v% in the qPCR reaction liquid, for example, 1 v/v%, 2 v/v%, 3 v/v%, 4 v/v%, or 5 v/v%, more preferably, a final concentration of 3 v/v%.
  • the tetramethylammonium chloride has a final concentration of 10-100 mM, preferably 20-80 mM in the qPCR reaction liquid, for example, 20 mM, 40 mM, 60 mM, or 80 mM, more preferably, a final concentration of 35 mM.
  • the present invention provides a qPCR reaction liquid, which contains the foregoing composition.
  • the qPCR reaction liquid further comprises a sample, for example, a nucleic acid-extracted sample and/or a non-nucleic acid-extracted sample.
  • the qPCR reaction liquid further comprises a primer and a probe for qPCR.
  • the qPCR reaction liquid further comprises a dNTP, a DNA polymerase, and a PCR buffer.
  • qPCR reaction liquid refers to a mixture capable of detecting a nucleic acid using fluorescence quantitative PCR.
  • the qPCR reaction liquid comprises the foregoing composition, a primer and a probe, a dNTP, a DNA polymerase, and a PCR buffer.
  • the present invention provides a use of the foregoing composition in improvement of qPCR detection performance, wherein advantageously, the improvement refers to improvement of performance of sensitivity, specificity, and/or interference resistance.
  • the term "detection performance” mainly refers to sensitivity, specificity, and interference resistance.
  • the present invention provides a method for preparing a qPCR reaction liquid, the method comprising a step of mixing a sample with a reaction buffer and the foregoing composition.
  • the reaction buffer comprises, for example, a dNTP, a DNA polymerase, and a PCR buffer. Further, a primer and a probe may be comprised.
  • the use of the composition of the present invention can improve the sensitivity, specificity, and interference resistance of fluorescence quantitative PCR, and the concentration of the composition of the present invention used can further improve the sensitivity, specificity, and interference resistance of the fluorescent quantitative PCR.
  • improving the detection performance can better provide molecular evidence for disease diagnosis and make adequate preparation for disease prevention and control, and can control infection sources for infectious and harmful infectious diseases in a timely manner, so as to block virus pandemics and outbreaks.
  • Example 1 Interference resistance performance of different compositions of the present invention in HCV detection
  • a substance such as SDS, bilirubin, and triglyceride, that causes PCR inhibition or interference is often added to a sample. Therefore, the investigation on the interference resistibility of PCR amplification reagents has become one of foci of performance of PCR reagents.
  • the composition of the present invention was applied to an HCV plasma sample with an interferent, and comparative detection was carried out with an HCV plasma sample without an interferent.
  • Comparative detection was performed on a low-concentration HCV sample (about 500 IU/mL) with an interference factor which was hemoglobin at a concentration of 2 g/dL.
  • the detection scheme was to repeat detection on the sample ten times under each condition, and compare the detection repeatability and the detection rate.
  • the detection method employed for the sample was a direct amplification method of a sample without nucleic acid extraction of 10 ⁇ L sample + 10 ⁇ L nucleic acid releasing agent + 30 ⁇ L PCR reaction liquid. Different combinations refer to combinations containing different compositions in a PCR reaction liquid.
  • the improvement of the detection capability by the compositions of the present invention differed to a certain extent among the low-concentration samples with the inhibitor.
  • the combination (Combination 8) containing all the ingredients all of the 10 low-concentration samples were detected.
  • the combination (Combination 9) not containing any component of the present invention none of the 10 samples was detected. Therefore, the composition of the present invention had obvious advantages regarding the inhibition resistibility in the detection of low-concentration HCV samples.
  • the different compositions of the present invention all had a positive effect on the improvement of the interference resistance.
  • Example 2 Interference resistance performance of different compositions of the present invention for detection of COVID-19 viruses
  • Comparative detection was performed on a low-concentration COVID-19 virus (hereinafter referred to as novel coronavirus or 2019-nCoV) nucleic acid sample (1000 copies/mL) with an interference factor.
  • the interference sample was a respiratory oropharyngeal swab sample with obvious turbid deposits.
  • the detection scheme was to repeat detection on the sample ten times under each condition, and compare the detection repeatability and the detection rate.
  • the detection method employed for the sample was an amplification method of 10 ⁇ L interference sample + 10 ⁇ L nucleic acid releasing agent + 1 ⁇ L novel coronavirus nucleic acid sample + 30 ⁇ L PCR reaction liquid, to investigate the influence on novel coronavirus nucleic acid amplification effects under different conditions.
  • Different combinations refer to combinations containing different compositions in a PCR reaction liquid. As shown in Table 3, "+” indicates that the component was added, and “-” indicates that the component was not added (after all ingredients were added to the qPCR reaction liquid, the ingredients had the following final concentrations: bovine serum albumin, 80 ⁇ g/mL; dithiothreitol, 3 mM; sorbitol, 4 w/v%; betaine, 0.8 mol/L; ammonium sulfate, 10 mM; formamide, 3v/v%; and tetramethylammonium chloride, 35 mM).
  • Control 1 an interference-free sample (replaced with TE), a PCR reaction liquid without additive components
  • Control 2 an interference-free sample (replaced with TE), a PCR reaction liquid with additive components.
  • Example 3 Use of the composition of the present invention in improvement of interference resistance performance of HCV detection
  • a composition of the present invention containing 7 components (bovine serum albumin, 80 ⁇ g/ml; dithiothreitol, 3 mM; sorbitol, 4 w/v%; betaine, 0.8 mol/L; ammonium sulfate, 10 mM; formamide, 3 v/v%; and tetramethylammonium chloride, 35 mM) was applied to an HCV plasma sample with an interferent, and comparative detection was carried out with an HCV plasma sample without an interferent.
  • 7 components bovine serum albumin, 80 ⁇ g/ml; dithiothreitol, 3 mM; sorbitol, 4 w/v%; betaine, 0.8 mol/L; ammonium sulfate, 10 mM; formamide, 3 v/v%; and tetramethylammonium chloride, 35 mM
  • a PCR amplification reagent without the composition of the present invention was used as a control component, and was also added to HCV plasma samples with an interferent and without an interferent for comparative detection.
  • this method employed direct sample amplification without nucleic acid extraction, the interference resistibility of the amplification reagent against the interference and inhibition effects in the sample was investigated in wider dimensions.
  • the composition was added to a PCR amplification reagent without the composition of the present invention, and a comparison test was carried out with a PCR amplification reagent without the composition of the present invention. Comparison of the measured Ct values of the samples was investigated. The Ct value was negatively correlated with the amplification efficiency, i.e., the larger the Ct value, the lower the amplification efficiency. Table 8.
  • the composition of the present invention had a significantly improved interference resistibility against common interferents in serum samples. No matter whether the sample was a low-concentration or a medium/high-concentration HCV sample, there was no significant difference in the amplification efficiency between the interference sample with an interference factor and the control sample without the interference factor.
  • the amplification efficiency was significantly decreased (the amplification Ct value was negatively correlated with the amplification efficiency, i.e., the larger the Ct value, the lower the amplification efficiency. No Ct means no amplification.).
  • the control reagent could not achieve the effect of an amplification test.
  • the low/medium-concentration HCV samples with lower concentrations none of the interference samples could be amplified.
  • This control experiment showed that the composition of the present invention had a significantly improved interference resistibility for qPCR amplification, especially in a sample lysis amplification technology that does not require nucleic acid extraction or purification.
  • Example 4 Use of the composition of the present invention in improvement of sensitivity of HCV detection
  • composition of the present invention containing all 7 ingredients (all the ingredients had the following final concentrations after being added to a qPCR reaction liquid: bovine serum albumin, 80 ⁇ g/mL; dithiothreitol, 3 mM; sorbitol, 4 w/v%; betaine, 0.8 mol/L; ammonium sulfate, 10 mM; formamide, 3 v/v%; and tetramethylammonium chloride, 35 mM).
  • bovine serum albumin 80 ⁇ g/mL
  • dithiothreitol 3 mM
  • sorbitol 4 w/v%
  • betaine 0.8 mol/L
  • ammonium sulfate 10 mM
  • formamide formamide
  • 3 v/v% tetramethylammonium chloride
  • the comparison method adopted was gradual gradient dilution of a clinically diagnosed positive hepatitis C virus (HCV) sample, i.e., 10-fold dilution (1:9, v/v), 100-fold dilution (1:99, v/v), and 1000-fold dilution (1:999, v/v).
  • HCV hepatitis C virus
  • a comparative test was carried out by using 10 ⁇ L nucleic acid releasing agent + 10 ⁇ L HCV sample + 30 ⁇ L PCR reaction liquid.
  • the real-time fluorescent quantitative PCR (Real-time qPCR) amplification test procedure was as shown in Table 9: Table 9.
  • Amplification test procedure for HCV samples employed in the present invention Step Temperature Time Number of cycles Reverse transcription 50°C 30 min 1 Pre-denaturation 95°C 1 min 1 Denaturation 95°C 15 sec 45 Annealing, extension, and fluorescence collection 60°C 30 sec
  • the comparison results in Table 10 showed that for the composition of the present invention in the PCR amplification system of the present invention, the sensitivity aspect of the RT-PCR was significantly improved.
  • the detection capability of the kit was negatively correlated with the cycle threathold (Ct) value. That is, the smaller the Ct value at the same concentration, the higher the detection capability; the larger the Ct value, the lower the detection capability. No Ct means no amplification.
  • the composition of the present invention had a significant improvement in the detection capability for nucleic acids.
  • the composition had a significantly improved detection capability for low-concentration nucleic acids as compared with the results of the control groups for the composition of the present invention, and had a significantly improved detection capability for clinically low-concentration samples.
  • PCR additive components in the present invention could significantly improve the sensitivity and interference resistance of hepatitis C virus (HCV) detection, and could achieve timely and rapid diagnosis of diseases.
  • Table 10 Effects of the composition of the present invention on Ct values of nucleic acid amplification PCR reaction liquid with the composition of the present invention
  • Commercial PCR reaction liquid HCV sample 26.64 28.09 HCV sample diluted 10x 29.89 32.21 HCV sample diluted 100x 33.11 35.83 HCV sample diluted 1000x 36.32 No Ct
  • Example 5 Use of the composition of the present invention in improvement of interference resistance performance of novel coronavirus detection
  • composition of the present invention containing all 7 ingredients (after all the ingredients were added to a qPCR reaction liquid, the ingredients had the following final concentrations: bovine serum albumin, 80 ⁇ g/mL; dithiothreitol, 3 mM; sorbitol, 4 w/v%; betaine, 0.8 mol/L; ammonium sulfate, 10 mM; formamide, 3 v/v%; and tetramethylammonium chloride, 35 mM).
  • the comparison scheme was to add 1 ⁇ L of a novel coronavirus nucleic acid at a concentration of about 1000 copies/mL into PCR Mastermix prepared, and at the same time add 10 ⁇ L of a sample with an inhibition effect and 10 ⁇ L of a nucleic acid releasing agent, to construct a reaction system with a total volume of 50 ⁇ L, so as to verify the interference resistibility of the novel coronavirus detection system in the amplification system.
  • Example 6 Use of the composition of the present invention in improvement of sensitivity of novel coronavirus detection
  • the ingredients had the following final concentrations: bovine serum albumin, 80 ⁇ g/mL; dithiothreitol, 3 mM; sorbitol, 4 w/v%; betaine, 0.8 mol/L; ammonium sulfate, 10 mM; formamide, 3 v/v%; and tetramethylammonium chloride, 35 mM).
  • bovine serum albumin 80 ⁇ g/mL
  • dithiothreitol 3 mM
  • sorbitol 4 w/v%
  • betaine 0.8 mol/L
  • ammonium sulfate 10 mM
  • formamide formamide
  • 3 v/v% tetramethylammonium chloride
  • the comparison method adopted was gradual gradient dilution of a clinically diagnosed positive novel coronavirus (2019-nCoV) nucleic acid sample, i.e., 10-fold dilution (1:9, v/v), 100-fold dilution (1:99, v/v), 1000-fold dilution (1:999, v/v), and 10000-fold dilution (1:9999, v/v).
  • the comparative test was carried out by using 45 ⁇ L PCR reaction liquid + 5 ⁇ L nucleic acid sample.
  • the real-time fluorescent quantitative PCR (Real-time qPCR) amplification test procedure was as shown in Table 13: Table 13.
  • Amplification test procedure for 2019-nCoV nucleic acid employed in the present invention Step Temperature Time Number of cycles Reverse transcription 50°C 30 min 1 Pre-denaturation 95°C 1 min 1 Denaturation 95°C 15 sec 45 Annealing, extension, and fluorescence collection 60°C 30 sec
  • the comparison results in Table 14 showed that for the composition of the present invention in the PCR amplification system of the present invention, the sensitivity of RT-PCR was significantly improved.
  • the detection capability of the kit was negatively correlated with the cycle threathold (Ct) value. That is, the smaller the Ct value at the same concentration, the higher the detection capability; the larger the Ct value, the lower the detection capability. No Ct means no amplification.
  • the composition of the present invention had a significant improvement in the detection capability for nucleic acids.
  • the composition had a significantly improved detection capability for low-concentration nucleic acids as compared with the results of the control groups for the composition of the present invention, and had a significantly improved detection capability for clinically low-concentration samples. Table 14.
  • Example 7 Use of the composition of the present invention in improvement of detection specificity
  • composition of the present invention containing 7 components (after all the ingredients were added to a qPCR reaction liquid, the ingredients had the following final concentrations: bovine serum albumin, 80 ⁇ g/mL; dithiothreitol, 3 mM; sorbitol, 4 w/v%; betaine, 0.8 mol/L; ammonium sulfate, 10 mM; formamide, 3 v/v%; and tetramethylammonium chloride, 35 mM).
  • bovine serum albumin 80 ⁇ g/mL
  • dithiothreitol 3 mM
  • sorbitol 4 w/v%
  • betaine 0.8 mol/L
  • ammonium sulfate 10 mM
  • formamide formamide
  • 3 v/v% tetramethylammonium chloride
  • the composition of the present invention was applied to analysis of human genome polymorphism (human APOE gene rs7412) for specificity comparison of amplification tests.
  • the experimental design was as follows: Table 15.

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Claims (10)

  1. Composition pour améliorer la performance de détection de qPCR, la composition comprenant : albumine de sérum bovin, sorbitol, sulfate d'ammonium, formamide et chlorure de tétraméthylammonium, et au moins un parmi le dithiothréitol et la bétaïne, dans laquelle les composants de la composition sont formulés pour avoir les concentrations finales suivantes après avoir été ajoutés à un liquide de réaction qPCR : protéine de sérum bovin, 10-150 µg/ml ; dithiothréitol, 1-10 mM ; sorbitol, 1-10 w/v%, bétaïne, 0,5-4 mol/L ; sulfate d'ammonium, 2-50 mM ; formamide, 0,1-10 v/v% ; et chlorure de tétraméthylammonium, 10-100 mM.
  2. Composition selon la revendication 1, dans laquelle la composition comprend de l'albumine de sérum bovin, du dithiothréitol, du sorbitol, du sulfate d'ammonium, du formamide et du chlorure de tétraméthylammonium.
  3. Composition selon la revendication 1, dans laquelle la composition comprend de l'albumine de sérum bovin, du sorbitol, de la bétaïne, du sulfate d'ammonium, du formamide et du chlorure de tétraméthylammonium.
  4. Composition selon la revendication 1, dans laquelle la composition comprend de l'albumine de sérum bovin, du dithiothréitol, du sorbitol, de la bétaïne, du sulfate d'ammonium, du formamide et du chlorure de tétraméthylammonium.
  5. Composition selon la revendication 4, dans laquelle les composants de la composition sont formulés pour avoir les concentrations finales suivantes après avoir été ajoutés au liquide de réaction de qPCR : protéine de sérum bovin, 80-120 µg/ml ; dithiothréitol, 2-8 mM ; sorbitol, 4-6 w/v% ; bétaïne, 0,6-1 mol/L ; sulfate d'ammonium, 8-15 mM ; formamide, 0,5-5 v/v% ; et chlorure de tétraméthylammonium, 20-80 mM.
  6. Composition selon la revendication 4, dans laquelle les composants de la composition sont formulés pour avoir les concentrations finales suivantes après avoir été ajoutés au liquide de réaction de qPCR : albumine de sérum bovin, 80 µg/mL ; dithiothréitol, 3 mM ; sorbitol, 4 w/v% ; bétaïne, 0,8 mol/L ; sulfate d'ammonium, 10 mM ; formamide, 3 v/v% ; et chlorure de tétraméthylammonium, 35 mM.
  7. Liquide de réaction de qPCR, comprenant la composition de l'une des revendications 1 à 6.
  8. Liquide de réaction de qPCR selon la revendication 7, comprenant en outre un échantillon, par exemple un échantillon extrait de l'acide nucléique et/ou un échantillon non extrait de l'acide nucléique.
  9. Utilisation de la composition selon l'une des revendications 1 à 6 dans l'amélioration de la performance de détection de qPCR, où avantageusement, l'amélioration fait référence à l'amélioration de la performance de la sensibilité, de la spécificité, et/ou de la résistance aux interférences.
  10. Méthode de préparation d'un liquide de réaction de qPCR, comprenant une étape de mélange d'un échantillon avec un tampon de réaction et la composition selon l'une des revendications 1 à 6.
EP20931793.2A 2020-04-23 2020-10-15 Composition pour améliorer la performance de test pcr en temps réel, liquide de réaction, utilisation et procédé Active EP4006167B1 (fr)

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CN202010326989.0A CN111218502B (zh) 2020-04-23 2020-04-23 提升qPCR检测性能的组合物、反应液、用途及方法
PCT/CN2020/121056 WO2021212771A1 (fr) 2020-04-23 2020-10-15 Composition pour améliorer la performance de test pcr en temps réel, liquide de réaction, utilisation et procédé

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CN116179656B (zh) * 2022-12-09 2024-04-09 南京诺唯赞生物科技股份有限公司 一种qPCR扩增反应液及用途
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CN108728518A (zh) * 2017-03-31 2018-11-02 广东顺德工业设计研究院(广东顺德创新设计研究院) 实时荧光定量pcr探针检测法及其反应液和试剂盒
CN110945142A (zh) * 2017-06-20 2020-03-31 乌利赛生物医学股份公司 通过聚合酶链式反应直接在原始样品中检测目标dna并通过高分辨熔解分析进行基因分型的方法
CN108570498B (zh) * 2018-05-25 2021-11-23 山东维真生物科技有限公司 用于检测人类cyp2c9和vkorc1基因多态性的引物探针组合物、试剂盒及应用
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CN110527747B (zh) * 2019-08-01 2023-01-10 北京农学院 一种检测猪瘟病毒野毒株的试剂盒
CN111218502B (zh) * 2020-04-23 2020-07-21 圣湘生物科技股份有限公司 提升qPCR检测性能的组合物、反应液、用途及方法

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EP4006167A4 (fr) 2022-11-30
EP4006167C0 (fr) 2023-11-29
CN111218502B (zh) 2020-07-21
WO2021212771A1 (fr) 2021-10-28
CN111218502A (zh) 2020-06-02
ZA202212648B (en) 2023-03-29
EP4006167A1 (fr) 2022-06-01
US20230193369A1 (en) 2023-06-22
BR112022021338A2 (pt) 2022-12-13

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